Elsevier

DNA Repair

Volume 8, Issue 10, 2 October 2009, Pages 1242-1249
DNA Repair

Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity

https://doi.org/10.1016/j.dnarep.2009.07.008Get rights and content

Abstract

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle's high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity.

Introduction

Mitochondrial DNA (mtDNA) incurs multiple forms of damage from both physiological and environmental sources. Since the presence of the mitochondrial genome is vital for eukaryotic viability, processes that maintain the integrity of mtDNA can likewise have global effects on cellular stability. The importance of mtDNA is underpinned by the identification of mtDNA mutations which result in a number of known genetic diseases. Additionally, the accumulation of mtDNA damage has been proposed to contribute to aging and age-related disorders, underscoring the importance of mtDNA maintenance for normal cellular function [1].

Progress in this field is revealing several pathways that are responsible for maintenance of the mitochondrial genome. Mitochondrial base excision repair (mtBER) and the proofreading 3′-5′ exonuclease activity of the mitochondrial polymerase, Pol γ, are known to impact mtDNA maintenance [2], [3], [4]. In addition, recent work has begun to elucidate other possible mechanisms for processing mtDNA lesions, including translesion DNA synthesis and double-strand break repair [5], [6], [7], [8], [9].

Of these multiple repair pathways, mtBER may be particularly significant. MtDNA is thought to be constantly bombarded with reactive oxygen species (ROS) due to its close proximity to the electron transport chain [10]. Studies have indicated that the mtBER pathway is largely responsible for repairing the oxidative damage to mtDNA [2], [11], [12]. Until recently, short-patch (SP) mtBER was the only accepted mtBER pathway, in which damaged nucleotides are replaced one at a time. There is now substantial evidence for long-patch (LP) BER in the mitochondrial compartment [13], [14], [15], although the proteins involved in this pathway are disputed.

Analysis of yeast and mammalian nuclear BER indicates that the FEN1 flap endonuclease is responsible for cleavage of the 5′ flap produced by LP BER [16]. The FEN1 family of proteins are evolutionarily conserved from archeabacteria to humans and are involved in DNA replication and repair [17], [18], [19]. The Saccharomyces cerevisiae FEN1 ortholog, Rad27p, plays an integral role in cleavage of 5′ DNA flaps created during Okazaki fragment processing [20], [21], [22], [23], [24], the processing of intermediates during nuclear LP BER [16], [25], prevention of sequence duplications and repeat sequence expansions [17], [25], [26], [27], [28], [29], [30], as well as being implicated in double-strand break repair [18], [27], [31].

In contrast to the roles of FEN1 in nuclear DNA maintenance, the full extent of FEN1 utilization in the mitochondria is unclear. Recent studies demonstrated that a 5′ flap removal activity exists in mitochondrial extracts of HeLa [13] and HCT116 [15] cell lines; however, these authors concluded that this activity was not due to FEN1. In contrast, another study identified FEN1 in mitochondrial extracts and concluded that LP mtBER is strongly dependent on FEN1 activity [14]. Although these studies provide clear evidence for LP mtBER, they differ in the enzyme proposed to cleave the 5′ flaps. Here we use genetic and physiological analysis to examine the role of the yeast flap endonuclease Rad27p in mtDNA maintenance. We demonstrate, for the first time, that deletion of RAD27 impacts the frequency of multiple types of mtDNA mutations, suggesting direct involvement in mtDNA repair. Additionally, we show evidence for Rad27p localization to yeast mitochondria through fractionation and fluorescence microscopy. Finally, we show evidence for mitochondrial localization of FEN1 in different types of mouse tissue, suggesting that its role in mtDNA maintenance is functionally conserved. These studies support a role for Rad27p/FEN1 in LP BER of mtDNA.

Section snippets

Deletion of RAD27 results in mitochondrial DNA mutations

The mitochondrial genome encodes some of the components of the electron transport chain (ETC) as well as the tRNAs and rRNAs required for their translation. Specific point mutations, deletions, rearrangements, or complete loss of the mitochondrial genome may all impair the function of the ETC and lead to cellular respiration deficiency. To determine if deletion of RAD27 caused mitochondrial genome instability, we measured the frequency of respiration loss, which is a functional correlate of

Discussion

FEN1 activity is critical for the normal maintenance of nuclear DNA. This enzyme has demonstrated roles in genome replication, DNA repair, and recombination. A role for FEN1 in mtDNA maintenance has only recently been suggested [14]; however, the mitochondrial subcellular localization of FEN1 has been debated [13], [14], [15]. Here we corroborate the findings of Liu et al. [14] with our genetic analysis of RAD27 deletion strains, and validate the localization of FEN1 in yeast and mice. We have

Growth media and strains

All growth media used in this study were previously described [6]. All S. cerevisiae strains used in this study (Table 1) are isogenic with DFS188 (MATa ura3-52 leu2-3, 112 lys2 his3 arg8∷hisG; ρ+), a derivative of D273-10B, except EAS736 and EAS738, which are derived from DFS160 (MATα ade2-101 leu2Δ ura3-52 arg8-Δ∷URA3 kar1-1) [54]. The rad27-Δ strain was constructed by one-step gene transplacement of the wild-type gene with the kanMX marker using standard methods [55]. The RAD27-citrine-3HA

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

Our research was supported by US National Science Foundation grant MCB0543084 (E.A.S.), US National Institutes of Health grants HL-33333 (S-S.S.) and 1 F31 GM078700 (L.K.). We are grateful to Dr. Robert Maul and Dr. Shona Mookerjee for critical reading of the manuscript and helpful comments and Dr. Thomas D. Fox for the gift of the anti-Cit1p antibody.

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